1. Field of the Invention
This invention resides in the field of reference controls for analytical determinations of protein levels in human bodily fluids. More specifically, this invention relates to reference controls for C-reactive protein determinations.
2. Description of the Prior Art
Cardiovascular disease is the leading cause of death worldwide. Presently, the only widely accepted indicator of a risk of an adverse cardiovascular event is the cholesterol level, and yet half of all cardiovascular events occur in people with normal plasma lipid levels.
C-reactive protein (hereinafter referred to as xe2x80x9cCRPxe2x80x9d) is a plasma protein that is synthesized in the liver and consists of five identical, non-glycosylated polypeptide subunits that are noncovalently linked to form a disc-shaped pentamer with a molecular weight of about 125,000. CRP is synthesized by hepatocytes in response to cytokines released into the liver by activated leukocytes. CRP is a nonspecific marker of inflammation and its level increases as a result of tissue injury or infection, elevating rapidly within 4 to 6 hours of the onset of acute levels of these conditions. CRP may rise to 25-35 mg/L after surgery, and peak at 30-35 mg/L during acute bacterial infection. In situations of severe trauma, the CRP concentration in plasma rises to 500-1,000 mg/L.
CRP is also a risk indicator for coronary heart disease. Among the various prognostic markers of heart disease, such as serum amyloid A, soluble intercellular adhesion molecule type 1, interleukin-6, homocysteine, total cholesterol, LDL, apolipoprotein B-100, HDL, and ratio of total cholesterol to HDL, CRP is the strongest predictor of cardiovascular events. When apparently healthy adults are tested for CRP, the fourth quartile (upper 25%) of those tested have been shown to have over four times the risk of those in the first quartile (with a confidence level of 95%), a ratio significantly greater than those of each of the markers listed above.
The use of CRP as a predictor of cardiovascular events differs however from its use as a detector of other types of inflammation or tissue injury, or infection, since the increases that indicate a risk of coronary heart disease are significantly lower than the increases associated with other conditions. For coronary heart disease predictions, therefore, high-sensitivity CRP (xe2x80x9chsCRPxe2x80x9d) detection methods are used. Automated analyzers on which tests for hsCRP can be performed include Dade Behring BN II Plasma Protein System (Dade Behring, Incorporated, Deerfield, Ill., USA), Abbott Laboratories IMx Immunoassay Analyzer (Abbott Laboratories, Abbott Park, Ill., USA), IMMULITE (Diagnostics Products Corporation, Los Angeles, Calif., USA), and IMMAGE (Beckman Coulter, Inc., Fullerton, Calif., USA). The Dade Behring BN II assay utilizes a monoclonal antibody on a polystyrene particle with fixed-time nephelometric measurements. The Abbott IMx assay is a two-site chemiluminescent enzyme immunometric assay with one monoclonal and one polyclonal anti-CRP antibody. The Beckman Coulter IMMAGE assay uses a polyclonal anti-CRP antibody on latex particles with rate nephelometric measurements. The detection limits for these assays range from 0.01 mg/L to 1.0 mg/L, and these instruments must be calibrated for accuracy at CRP concentrations within these ranges, which are below those traditionally measured in clinical laboratories for less sensitive CRP assays.
Because of the high sensitivity of hsCRP assays and the analytical variances that are typically encountered, quality control is important in monitoring the precision and accuracy of the assays and assay instruments. Reference controls are obtained in two ways. In the first, plasma or serum from blood donors is screened to identify units containing suitably low levels of CRP, and the units thus identified are pooled. In the second, CRP is removed from plasma or serum by affinity chromatography using anti-CRP antibodies. Purified CRP is then added to the resulting CRP-depleted base matrix to achieve the particular levels desired. Both of these methods are time-consuming and costly. Screening, for example, requires extensive testing and may not produce units with the low levels desired, or may not yield the volume needed. Selective removal of CRP by affinity chromatography requires separation techniques such as dialysis and chromatography, following by concentration.
It has now been discovered that low-concentration CRP reference controls can be prepared from higher-level starting materials by simple filtration using silica-type filter media. Biological fluids obtained from normal healthy individuals can be used as the starting materials. This procedure avoids the need for antibodies to capture the protein, and instead uses filter media that have traditionally been used for removing lipids. Surprisingly, the removal of CRP by the silica-type filter media is selective toward CRP, having little or no effect on other proteins in the starting matrix, and accordingly little or negligible change in the total protein content. Notably, the immunoglobulin content is not significantly affected by the filtration, nor are albumins.
Low-level preparations obtained in this manner can be blended with higher-level preparations such as further quantities of the starting material that have not been filtered, to achieve reference controls with intermediate levels of CRP. By preparing blends in a series of different proportions, multiple controls can be obtained that will serve as materials for an accurate control of the assay and instrument over a range encompassing the anticipated sample results. Controls of particular interest in this invention are those with CRP concentrations below the medical decision point, and most particularly those of CRP concentrations that are less than or equal to 1.0 mg/L.
Details of these and other features, embodiments, and advantages of the invention will become apparent from the description that follows.